Summary of work: Werner's Syndrome (WS) is a homozygous recessive disease characterized by early onset of many characteristics of normal aging, such as wrinkling of the skin, graying of the hair, cataracts, diabetes, and osteoporosis. The symptoms of WS begin to appear around the age of puberty, and most patients die before age 50. Because of the acceleration of aging in WS, the study of this disease will hopefully shed light on the degenerative processes that occur in normal aging. Cells from WS patients grow more slowly and senescence at an earlier population doubling than age-matched normal cells, possibly because these cells appear to lose the telomeric ends of their chromosomes at an accelerated rate. In general, WS cells have a high level of genomic instability, with increased amounts of DNA deletions, insertions, and rearrangements. These effects could potentially be the result of defects in DNA repair, replication, and/or recombination, although the actual biochemical defect remains unknown. The gene that is defective in WS, the WRN gene, has recently been identified and characterized as a helicase. Thus, the genetic evidence also points to a role for the WRN protein in some aspect of DNA metabolism. We are using several avenues to identify and characterize the biochemical defect in WS cells. One approach is to compare the DNA metabolic activities of normal and WS cells. After treatment with certain DNA damaging agents, both the cellular sensitivity and levels of overall DNA repair in WS cells is not elevated. However, WS cells appear to have a subtle defect in transcription-coupled repair, the highly efficient removal of lesions from the transcribed strand of active genes. Moreover, a survey of overall transcription in cells from premature aging syndromes indicated that WS cells have lower transcription rates than in normal cells in vivo. Experiments with cell extracts suggest that this decrease in transcription might be due to the presence of an inhibitor of RNA polymerase II in WS cells. Other studies are aimed at determining whether this shortfall in total transcription might represent lower transcription of particular subsets of genes. The observed inhibition of RNA polymerase II transcription might also explain the loss of transcription-coupled repair. Studies regarding the transcription and repair activities of WS cells are ongoing; in particular, the establishment of a WS cell line transfected with the normal WRN gene will allow us to assess whether these repair and transcription problems are manifestations of the primary molecular defect in WS. We are now making purified WRN protein for use in a number of basic and complex biochemical assays. Thus far, the WRN gene has been inserted into a baculovirus vector that has been transfected into insect cells, which will putatively allow overproduction and subsequent purification of significant quantities of WRN protein. Once purified to homogeneity, WRN protein will be examined for its biochemical activities and potential interactions with other proteins. Our ongoing and future studies will be directed towards elucidation of the causes of the accelerated aging phenotype in WS, with hope that this knowledge can also be applied to our current understanding of both the aging of cells and organisms in general.